由於內耳特殊之結構及位置，如何有效地將藥物等輸送至內耳為一大考驗。在內耳的醫學領域中，噪音性聽損為常見的聽力相關病症，且有相當高比例之患者接受化療藥物治療後，產生耳毒性等副作用；耳毒性和耳蝸內氧化壓力有關，活性氧物質會破壞DNA及蛋白質，從而導致細胞損傷、壞死和凋亡等不可逆傷害。siRNA為新興醫學療法，其應用於基因敲落為相當重要之技術，在不同的醫學領域中針對標靶基因給予細胞治療甚至再生之效用。本研究探討siRNA吸附溶菌酶微氣泡併超音波介導技術，延伸於內耳領域基因治療，以獲得安全且有效之基因敲落，嘗試發展出內耳之創新療法。
以正負電荷吸附之LyzMBs-siRNA透過動態光散射儀測量粒徑與電位，並進行細胞轉染實驗 (n=5)，其實驗組別分別為 (1) Control (no treatment)、(2) 單純siRNA溶液 (siRNA) 、(3) 施打超音波能量於siRNA溶液 (US+siRNA)、(4) siRNA溶液混合溶菌酶微氣泡 (LyzMBs+siRNA)、(5) 施打超音波能量於溶菌酶微氣泡混合siRNA溶液 (US+LyzMBs+siRNA)、(6) 吸附siRNA之溶菌酶微氣泡 (LyzMBs-siRNA)、(7) 施打超音波能量於吸附siRNA之溶菌酶微氣泡 (US+ LyzMBs-siRNA)。
根據實驗結果，單純溶菌酶微氣泡與吸附siRNA Cy3之溶菌酶微氣泡粒徑大小依序為 2.6 ± 0.08 µm、4.0 ± 0.49 µm；表面電位依序為 54.4 ± 2.70 mV、47.1 ± 1.27 mV；溶菌酶微氣泡吸附siRNA Cy3之吸附率為 65%；在體外試驗中，吸附款之溶菌酶微氣泡施打超音波後轉染效率提升至 48.18%。siRNA由於本身微負電結構，難以穿過帶負電之細胞膜，藉由吸附溶菌酶微氣泡結合超音波介導技術，溶菌酶微氣泡作為藥物載體，施打超音波造成微氣泡穴蝕效應，開啟細胞膜通透度，成功的將siRNA送入細胞、提高轉染效率並保護siRNA免受血清等降解。本研究將以超音波結合微氣泡對比劑之技術，探討溶菌酶微氣泡作為基因載體於內耳細胞進行基因敲落之可行性。

Since the special structure and position of the inner ear, how to effectively deliver drugs into the inner ear is a big challenge. Noise-induced hearing loss is a common hearing-related disorder, and there is a high percentage of patients receiving chemotherapy drugs have side effects such as ototoxicity. Ototoxicity is associated with increased cochlear oxidative stress. These reactive oxygen species can damage DNA and proteins, leading to irreversible damage such as cell damage, necrosis, and apoptosis. siRNA transfection is a new therapeutic method, and its application in gene knockdown is a very important technology. In different medical research fields, siRNA for specific target genes can provide therapeutic and even cell regeneration effects. This thesis aims to explore newly synthesized siRNA-coated lysozyme microbubble complexes with ultrasound-mediation, which extends gene therapy in the inner ear to obtain a safer and more effective method of gene knockdown, and try to develop innovative therapies for the inner ear.
The size distribution and Zeta potential of the siRNA-shelled LyzMBs in suspension were measured by DLS respectively. The in vitro (n=5) experimental parameters will be divided into seven groups: (1) no treatment (Control), (2) siRNA solution (siRNA) alone, (3) US combines with siRNA solution (US+siRNA), (4) siRNA solution mixed with LyzMBs (LyzMBs+siRNA), (5) US combined with LyzMBs and penetrating siRNA (US+LyzMBs+siRNA), (6) siRNA-shelled LyzMBs (LyzMBs-siRNA) alone; (7) US combined with LyzMBs-siRNA (US+LyzMBs-siRNA).
According to the results, siRNA grafted with LyzMBs (mean diameter of 2.6 ± 0.08 µm) were synthesized into complexes of LyzMBs-siRNA that had mean diameters of 4.0 ± 0.49 µm. The Zeta potentials of LyzMBs and LyzMBs-siRNA were 54.4 ± 2.70 mV and 47.1 ± 1.27 mV, respectively. The adsorption efficiency of siRNA Cy3 on cationic LyzMBs was 65%. In the in vitro study, US+LyzMBs-siRNA had the best transfection efficiency of 48.18 %. Positive charged polyplexes facilitate the cellular uptake by interaction with negative charged cell membrane. Ultrasonic application can cause microbubbles cavitation effect, enhance cell membrane permeability, deliver siRNA into cells, improve transfection efficiency and protect siRNA from degradation by serum. This study will investigate the role and safety of ultrasound-microbubble in the application of cochlear gene knockdown to explore the feasibility of lysozyme microbubbles as gene carriers in the inner ear.

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